重组人骨形态发生蛋白-2壳聚糖纳米微球的制备及检测

Preparation of rh-BMP-2 Loaded Chitosan Nanospheres and Its Properties in Vitro

目的制备负载重组人骨形态发生蛋白-2(rhBMP-2)壳聚糖纳米微球,并检测其粒径、形态、降解及药理特性,以评估壳聚糖纳米微球作为rhBMP-2缓释载体的可行性。方法以壳聚糖为原料、三聚磷酸钠为交联剂,通过离子交联法制备负载rhBMP-2壳聚糖纳米微球,应用透视电镜观察微球的形态、激光粒径,分析其粒径分布、溶菌酶降解,了解降解特性。通过酶联免疫吸附实验(ELISA)检测rhBMP-2壳聚糖微球的载药率、包封率和释药规律。结果离子交联法制备的壳聚糖纳米微球,平均粒径大小为230nm,成球性较好,包封率和载药率分别为(66.867±4.575)%、(33.437±2.290)μg/mg;体外释药试验rhBMP-2可以从壳聚糖纳米微球中缓慢释放,释放行为符合双向动力学规律,整个释放过程可达30 d。结论离子交联法可成功制备壳聚糖纳米微球并具有缓释rhBMP-2的能力,为进一步应用于骨组织工程研究提供实验依据。

Objective To prepare a sustained release system for rhBMP-2 and to evaluate the grain size, appearance, degradation, pharmaco-characteristic and the feasibility of chitosan nanospheres as a carrier for sustained release of rhBMP-2. Methods The rhBMP-2 loaded chitosan nanospheres were prepared using an ionic crosslinking method with tripolyphosphate （TPP） and chitosan. Transmission electron microscope was used to evaluate the morphological properties, and laser particle size analyzer was used to analyze particle size distribution. Lysozyme degradation experiment was performed to assess the biodegradation behavior. ELISA assay was carried out to determine the drug loading rate, entrapment efficiency and in vitro drug release kinetics. Results The rhBMP-2 loaded chitosan nanospheres was spherical in shape, and showed a smooth surface. The mean diameter was 230 nm. The average drug loading rate and encapsulation efficiency were （66.867±4.575）% and （33.437±2.290）μg/mg, respectively. The release experiment in vitro showed that the releasing property of rhBMP-2 loaded chitosan nanospheres was consistent with the two phase kinetic regulation and rhBMP-2 was controlled to release from the chitosan nanospheres over 30 days. Conclusion The rhBMP-2 loaded chitosan nanospheres prepared by ionic cresshnking method shows a good controlled release property. Such a novel type of controlled release system has the potential of being applied for the bone tissue engineering.